W-band pulsed EPR: a) probing 17O ligands in the Mo(V) site of sulfite oxidases; b) ascertaining the long distance limit in Pulse Dipolar Spectroscopy of Gd(III) labeled macrobiomolecules.
EMSL Project ID
44607
Abstract
Abstract W-band pulsed EPR: a) probing 17O ligands in the Mo(V) site of sulfite oxidases; b) ascertaining the long distance limit in Pulse Dipolar Spectroscopy of Gd(III) labeled macrobiomolecules. Principal Investigator: John H. Enemark (University of Arizona) The newly built unique W-band pulsed EPR spectrometer of EMSL enables Electron Spin Echo Modulation Spectroscopy to be extended to biological and environmental applications that were previously impossible. A long-standing problem in molybdenum enzymes (which are essential in all forms of life for the metabolism of carbon, sulfur and nitrogen compounds) is the structure of the catalytic Mo(V) site and the nature of the directly coordinated oxygen atom that plays a key role in the catalytic mechanisms of such enzymes, including: sulfite oxidase; xanthine oxidase; DMSO reductase; and nitrate reductase. Assessment of the role of this oxygen atom that is directly bound to Mo requires measurements of the hyperfine and nuclear quadrupole interactions for 'strongly' coupled 17O ligands, which have not been possible previously. We plan to obtain unambiguous information on the magnetic resonance parameters (nuclear quadrupole and hyperfine interactions) of such 17O ligands in the Mo(V) centers of sulfite oxidases from vertebrates, plants and bacteria. Both wild type and mutant enzymes will be investigated using 17O (I = 5/2) enrichment, and the results will be related to structural variation of active site using extended DFT calculations, and to variation of catalytic activity. In a second area, Pulsed Dipolar Spectroscopy (PDS), we plan to double the distance that is currently accessible from dipolar coupling between spin-labels, so that the expected upper limit will be ~150 A. This optimistic prognosis is based upon the expected synergy of the properties of the ESML W-band pulsed EPR spectrometer and the properties of Gd(III)-based spin-labels, which will be used instead of standard nitroxide radicals. The successful development of extended distance measurements using Gd(III) spin-labels and pulsed W-band EPR will substantially increase the types of biomacromolecular objects and functions that can be routinely investigated by PDS methods.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2011-10-01
End Date
2012-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Klein EL, AV Astashkin, A Raitsimring, and JH Enemark. 2013. "Applications of pulsed EPR spectroscopy to structural studies of sulfite oxidizing enzymes." Coordination Chemistry Reviews 257(1):110-118. doi:http://dx.doi.org/10.1016/j.ccr.2012.05.038
Raitsimring A, AV Astashkin, JH Enemark, I Kaminker, D Goldfarb, ED Walter, Y Song, and TJ Meade. 2013. "Optimization of pulsed-DEER measurements for Gd-based labels: choice of operational frequencies, pulse durations and positions, and temperature." Applied Magnetic Resonance 44(6):649-670. doi: 10.1007/s00723-012-0434-6